Antimicrobial Use, Resistance, and Impact to the Pork Industry: Monthly Update (April '05)

June 14, 2005 — Wondwossen A. Gebreyes (ed. David Burch)

Report prepared for the National Pork Board

May 15, 2005 Raleigh, NC

INTRODUCTION

In the past one month, a number of scientific studies reported findings on antimicrobial use, resistant strains and association with virulence. The studies included various host species as well as geographic locations. Antimicrobial resistance is a multi-faceted problem that involves various animal species and humans. In addition, because of the fact that numerous risk factors beyond mere antimicrobial use may play a role in the emergence and dissemination of resistance in wide geographical areas (including foreign travel etc.), understanding the role of each component would be crucial in curbing the resistance problem in general. New mechanisms of resistance to fluoroquinolones, persistence of fluoroquinolone resistance in conventional and antibiotic-free farms and resistance of pathogens isolated from wild reservoirs have been described. A summary of the relevant articles that have a direct or indirect significance to pork industry in the past one month is summarized below.

PREVALENCE AND ANTIMICROBIAL RESISTANCE

FLUOROQUINOLONE-RESISTANT CAMPYLOBACTER ISOLATES FROM CONVENTIONAL AND ANTIBIOTIC-FREE CHICKEN PRODUCTS (PRICE ET AL., 2005). The study investigated the use of fluoroquinolone (FQ) and its discontinuation in some poultry productions and its association with detection of FQ resistant Campylobacter jejuni in retail poultry meat and on farms. The study reported multiple findings. One major finding was that FQ-resistant strains were detected on 17% of samples using unsupplemented media and on 40% using agar supplemented with 4 µg/mL CIP. The justification for using antibiotic containing selective plate was to not underestimate prevalence as mixed infections could occur. However, such methods could also overestimate the findings by mutation of susceptible strains to resistant once they are exposed to FQ containing plates. In addition, this approach is not an NCCLS approved method to test for susceptibility. The two conventional companies did not use antibiotics one year prior to this study. However, significant proportions of products from both of these companies carried FQ-resistant strains of Campylobacter. The data suggest that past FQ use may result in persistent resistant Campylobacter populations. The study is interesting as a preliminary comparison. It had a limitation in its scope as only few producers in one state included and thus can not be generalizable. Despite the limitations, it has important implications that antimicrobial resistance could persist in a population even if the use in animal production is banned. His may have important implication in the pork industry as well though FQ is not used in swine production and therapy in the U.S.

IDENTIFICATION AND SURVEILLANCE OF ANTIMICROBIAL RESISTANCE DISSEMINATION IN ANIMAL PRODUCTION (BYWATER RJ). This is a review by the Bywater Consultancy on the antimicrobial use, agriculture and impact of banning antimicrobial use for production purposes. The European Union (EU) has withdrawn as AGP some compounds that remain in use in the United Sates. This difference in availability allows comparisons to be made of antimicrobial resistance outcomes with and without use of an AGP. Such comparisons so far show little apparent measurable benefit to human health resulting from the EU removal of AGP, and there is evidence of increased use of therapeutic antibiotics in animals to treat an apparent increased incidence of clinical disease. Microbial risk assessments are important in judging quantitatively or qualitatively whether the risk of using a particular AGP is acceptable in terms of potential hazard to human health. Resistance surveillance is an essential part of such microbial risk assessments, but such surveillance should be carefully planned to avoid confounding factors that could invalidate any conclusions.

SALMONELLA IN DAIRY OPERATIONS IN THE UNITED STATES: PREVALENCE AND ANTIMICROBIAL DRUG SUSCEPTIBILITY (BLAU DM ET AL., 2005). A national study of U.S. dairy operations conducted between March and September 2002 was reported. One third of the herd with 7.3% of fecal prevalence for Salmonella was reported. Antimicrobial susceptibility was done for 16 antimicrobial agents. More than three-fourth of the isolates (83%) were pansusceptible. It was shown that relatively little antimicrobial resistance detected in dairy operations.

HIGH-LEVEL AMINOGLYCOSIDE RESISTANT ENTEROCOCCI ISOLATED FROM SWINE (JACKSON CR ET AL., 2005). In this study, aminoglycoside resistance among enterococci collected in 1999 and 2000 was investigated. High level of resistance to gentamicin (MIC > or =500 microg/ml), kanamycin (MIC > or =500 microg/ml), or streptomycin (MIC > or =1000 microg/ml) was detected. Eight aminoglycoside resistance genes were detected using PCR, most frequently ant(6)-Ia and aac(6')-Ii from Enterococcus faecium. Twenty-four per cent (45/187) of total high-level aminoglycoside-resistant isolates and 26% (4/15) of isolates resistant to high levels of all three antimicrobials were negative for all genes tested. These data suggest that enterococci isolated from swine contain diverse and possibly unidentified aminoglycoside resistance genes. Though enterococci may not be of direct clinical and food safety concern, they may act as important reservoirs of antimicrobial resistance factors.

ANTIMICROBIAL RESISTANCE PROFILES OF CAMPYLOBACTER JEJUNI ISOLATES FROM WILD BIRDS IN SWEDEN (WALDENSTROM J ET AL., 2005). To establish baseline MICs in Campylobacter jejuni isolates from an environmental reservoir, the resistance profiles of 10 antimicrobial substances were determined for 137 C. jejuni isolates from wild birds in Sweden. Observed MICs were generally low, with only low to moderate incidence of resistance to the tested compounds. One isolate, however, was resistant to nalidixic acid and ciprofloxacin, indicating that quinolone-resistant genotypes of C. jejuni have the potential to spread to wild bird hosts.

GENOTYPING AND MOLECULAR CHARACTERIZATION OF RESISTANCE DETERMINANTS

CLASS 1 INTEGRON-ASSOCIATED GENE CASSETTES IN SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR AGONA ISOLATED FROM PIG CARCASSES IN BRAZIL (MICHAEL GB ET AL., 2005). Two strains of multi-drug resistant Salmonella enterica subsp. enterica serovar Agona isolates from pig carcasses were investigated for antimicrobial resistance genes and their location with particular reference to the detection of class 1 integrons. Both S. Agona strains carried conjugative plasmids of approximately 150 kb which harboured all resistance genes detected in the respective isolates. One of the strains (S. Agona 23) was resistant to chloramphenicol by catA1, to tetracycline and minocycline by tet(B), and to sulphonamides by sul1. In addition, it harboured a streptomycin resistance gene strA and a class 1 integron with a new aadA variant designated aadA23, which mediates resistance to streptomycin and spectinomycin. The results of this study show that large conjugative multiresistance plasmids are present in S. Agona from pigs. Analysis of the class 1 integrons revealed the presence of new variants of resistance genes so far not detected in Salmonella isolates.

CHARACTERIZATION OF ANTIMICROBIAL RESISTANCE AND CLASS 1 INTEGRONS FOUND IN ESCHERICHIA COLI ISOLATES FROM HUMANS AND ANIMALS IN KOREA (KANG HY ET AL., 2005). Antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea were characterized. Commensal E. coli isolates from animals were highly resistant to commonly used antimicrobial agents such as tetracycline, sulfamethoxazole, streptomycin, ampicillin and carbenicillin. Integrons were most prevalent in commensal E. coli isolates from poultry (44%), followed by clinical isolates from humans (33%), commensal isolates from swine (23%) and humans (13%). Class 1 integrons were mostly located in conjugative plasmids. E. coli isolates carrying an identical cassette array were phylogenetically unrelated. The horizontal transfer of class 1 integrons through conjugative plasmids seems to be responsible for wide dissemination of a particular type of class 1 integron amongst genotypically diverse strains.

EVIDENCE FOR MULTIPLE-ANTIBIOTIC RESISTANCE IN CAMPYLOBACTER JEJUNI NOT MEDIATED BY CMEB OR CMEF (PUMBWE L ET AL., 2005). An efflux system, CmeABC, in Campylobacter jejuni was previously described, and a second efflux system, CmeDEF, has now been identified. The substrates of CmeDEF include ampicillin, ethidium bromide, acridine, sodium dodecyl sulfate (SDS), deoxycholate, triclosan, and cetrimide, but not ciprofloxacin or erythromycin. The findings of this study suggest the involvement of a non-CmeB or -CmeF efflux pump or reduced uptake conferring multiple-antibiotic resistance, which can be selected after exposure to a fluoroquinolone. This finding underscores the increasingly diverse mechanisms of resistance to fluoroquinolones similar to other antimicrobials.

IMPACT TO THE PORK INDUSTRY

Most of the studies reported in the past month focused on molecular characterization of important resistance determinants. Among the most interesting findings was the detection of fluoroquinolone resistance in antibiotic free intensive farms even after discontinuation of antibiotic use for more than one year. Though this study was done on poultry production it may have important implications to pork and other food animals industries and it could imply that multiple risk factors other than direct antibiotic use play a role in emergence and persistence of antimicrobial resistant strains. A smaller study in Sweden also showed that wild birds could be important reservoirs of fluoroquinolone resistant Campylobacter.

REFERENCES

Blau DM, McCluskey BJ, Ladely SR, Dargatz DA, Fedorka-Cray PJ, Ferris KE, Headrick ML. 2005. Salmonella in dairy operations in the United States: prevalence and antimicrobial drug susceptibility. J Food Prot. 2005 Apr;68(4):696-702.

Bywater RJ. 2005. Identification and surveillance of antimicrobial resistance dissemination in animal production. Poult Sci. 2005 Apr;84(4):644-8.

Jackson CR, Fedorka-Cray PJ, Barrett JB, Ladely SR. 2005. High-level aminoglycoside resistant enterococci isolated from swine. Epidemiol Infect. 2005 Apr;133(2):367-71.

Kang HY, Jeong YS, Oh JY, Tae SH, Choi CH, Moon DC, Lee WK, Lee YC, Seol SY, Cho DT, Lee JC. 2005. Characterization of antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea. J Antimicrob Chemother. 2005 May;55(5):639-44.

Michael GB, Cardoso M, Schwarz S. 2005. Class 1 integron-associated gene cassettes in Salmonella enterica subsp. enterica serovar Agona isolated from pig carcasses in Brazil. J Antimicrob Chemother. 2005 May;55(5):776-9.

Price LB, Johnson E, Vailes R, Silbergeld E. 2005. Fluoroquinolone-resistant campylobacter isolates from conventional and antibiotic-free chicken products. Environ Health Perspect. 2005 May;113(5):557-60.

Pumbwe L, Randall LP, Woodward MJ, Piddock LJ. 2005. Evidence for multiple-antibiotic resistance in Campylobacter jejuni not mediated by CmeB or CmeF. Antimicrob Agents Chemother. 2005 Apr;49(4):1289-93.

Waldenstrom J, Mevius D, Veldman K, Broman T, Hasselquist D, Olsen B. 2005. Antimicrobial resistance profiles of Campylobacter jejuni isolates from wild birds in Sweden. Appl Environ Microbiol. 2005 May;71(5):2438-41.

APPENDIX

Environ Health Perspect. 2005 May;113(5):557-60. Fluoroquinolone-resistant campylobacter isolates from conventional and antibiotic-free chicken products. Price LB, Johnson E, Vailes R, Silbergeld E. Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe Street, Suite W6114, Baltimore, MD 21205, USA. lprice@jhsph.edu

The use of fluoroquinolones (FQs) in poultry production is an important issue in public health today. In February 2002, two prominent U.S. poultry companies pledged to stop using FQs for flock-wide treatment. One year later, we began a survey of Campylobacter isolates on chicken products from these two companies and from two producers claiming total abstention from antibiotic use. Using both standard isolation methods and new methods modified to enhance detection of FQ-resistant Campylobacter, we compared rates of FQ-resistant Campylobacter among these products. Four major findings were drawn from this study: a) antibiotic-free brands were not more likely to be contaminated with Campylobacter; b) a high percentage of products from the two conventional brands were contaminated with FQ-resistant Campylobacter (43 and 96%); c) these conventional brands had significantly higher odds of carrying resistant strains compared with antibiotic-free products; and d) supplementing media with FQs increased the sensitivity of detecting FQ-resistant strains among mixed populations of Campylobacter, thus reducing a bias toward underestimating the prevalence of FQ-resistant Campylobacter on samples. These results suggest that FQ resistance may persist in the commercial poultry environment in the absence of FQ-selective pressure and that these strains contaminate a larger proportion of foods than reported previously.

Appl Environ Microbiol. 2005 May;71(5):2438-41.

Antimicrobial Resistance Profiles of Campylobacter jejuni Isolates from Wild Birds in Sweden.

Waldenstrom J, Mevius D, Veldman K, Broman T, Hasselquist D, Olsen B.

Central Institute for Animal Disease Control (CIDC-Lelystad), 8219PH Lelystad, The Netherlands. dik.mevius@wur.nl.

In order to determine the occurrence and frequency of resistant strains of the bacterium Campylobacter jejuni and to establish baseline MICs in isolates from an environmental reservoir, the resistance profiles of 10 antimicrobial substances were determined for 137 C. jejuni isolates from wild birds in Sweden. Observed MICs were generally low, with only low to moderate incidence of resistance to the tested compounds. One isolate, however, was resistant to nalidixic acid and ciprofloxacin, indicating that quinolone-resistant genotypes of C. jejuni have the potential to spread to wild bird hosts.

Poult Sci. 2005 Apr;84(4):644-8. Related Articles, Links

Identification and surveillance of antimicrobial resistance dissemination in animal production.

Bywater RJ. Bywater Consultancy, Little Common House, Clungunford, Shropshire, SY7 0PL, United Kingdom. rbywater@onetel.com

Antimicrobial resistance is a growing problem in human medicine, and concern has been expressed that use of antimicrobials in animals may be a contributing factor. Although the majority of human pathogens showing antibiotic resistance have no link with animals, the issue of animal use of antimicrobials remains controversial, particularly with respect to antibiotic growth promoters (AGP). The European Union (EU) has withdrawn as AGP some compounds that remain in use in the United Sates. This difference in availability allows comparisons to be made of antimicrobial resistance outcomes with and without use of an AGP. Such comparisons so far show little apparent measurable benefit to human health resulting from the EU removal of AGP, and there is evidence of increased use of therapeutic antibiotics in animals to treat an apparent increased incidence of clinical disease. Microbial risk assessments are important in judging quantitatively or qualitatively whether the risk of using a particular AGP is acceptable in terms of potential hazard to human health. Resistance surveillance is an essential part of such microbial risk assessments, but such surveillance should be carefully planned to avoid confounding factors that could invalidate any conclusions.

J Food Prot. 2005 Apr;68(4):696-702.

Salmonella in dairy operations in the United States: prevalence and antimicrobial drug susceptibility.

Blau DM, McCluskey BJ, Ladely SR, Dargatz DA, Fedorka-Cray PJ, Ferris KE, Headrick ML.

Centers for Epidemiology and Animal Health, Animal and Plant Health Inspection Service, US Department of Agriculture, Fort Collins, Colorado 80526-8117, USA.

Salmonella serotypes are important foodborne pathogens of humans that can be acquired through consumption of contaminated meat and dairy products. Salmonella infection also can be a significant animal health issue. As part of a national study of U.S. dairy operations conducted between March and September 2002, fecal samples were collected from representative cows in 97 dairy herds in 21 states and were cultured to determine the prevalence of Salmonella shedding. Salmonella was recovered from the feces of at least one cow in 30.9% of the herds. Overall, 7.3% of fecal samples were culture positive for Salmonella. The three most frequently recovered serotypes were Salmonella Meleagridis (24.1%), Salmonella Montevideo (11.9%), and Salmonella Typhimurium (9.9%). The susceptibilities of Salmonella isolates recovered were determined using a panel of 16 antimicrobial drugs. Salmonella isolates recovered from dairy cows had relatively little resistance to these antimicrobial agents; 83.0% of the isolates were susceptible to all antimicrobials tested. This study provides updated information on the prevalence and susceptibility patterns of Salmonella in dairy herds and on cow and herd characteristics. These data contribute to our understanding of the ecology of Salmonella in the dairy farm environment.

Epidemiol Infect. 2005 Apr;133(2):367-71.

High-level aminoglycoside resistant enterococci isolated from swine.

Jackson CR, Fedorka-Cray PJ, Barrett JB, Ladely SR.

United States Department of Agriculture, Agricultural Research Service, Antimicrobial Resistance Research Unit, USA. cjackson@saa.ars.usda.gov

Approximately 42% (187/444) of swine enterococci collected between the years 1999 and 2000 exhibited high-level resistance to gentamicin (MIC > or =500 microg/ml), kanamycin (MIC > or =500 microg/ml), or streptomycin (MIC > or =1000 microg/ml). Eight aminoglycoside resistance genes were detected using PCR, most frequently ant(6)-Ia and aac(6')-Ii from Enterococcus faecium. Twenty-four per cent (45/187) of total high-level aminoglycoside-resistant isolates and 26% (4/15) of isolates resistant to high levels of all three antimicrobials were negative for all genes tested. These data suggest that enterococci isolated from swine contain diverse and possibly unidentified aminoglycoside resistance genes.

Antimicrob Agents Chemother. 2005 Apr;49(4):1289-93.

Evidence for multiple-antibiotic resistance in Campylobacter jejuni not mediated by CmeB or CmeF.

Pumbwe L, Randall LP, Woodward MJ, Piddock LJ.

Antimicrobial Agents Research Group, Division of Infection and Immunity, University of Birmingham, Birmingham, B15 2TT, United Kingdom.

An efflux system, CmeABC, in Campylobacter jejuni was previously described, and a second efflux system, CmeDEF, has now been identified. The substrates of CmeDEF include ampicillin, ethidium bromide, acridine, sodium dodecyl sulfate (SDS), deoxycholate, triclosan, and cetrimide, but not ciprofloxacin or erythromycin. C. jejuni NCTC11168 and two efflux pump knockout strains, cmeB::Kan(r) and cmeF::Kan(r), were exposed to 0.5 to 1 microg of ciprofloxacin/ml in agar plates. All mutants arising from NCTC11168 were resistant to ciprofloxacin but not to other agents and contained a mutation resulting in the replacement of threonine 86 with isoleucine in the quinolone resistance-determining region of GyrA. Mutants with two distinct phenotypes were selected from the efflux pump knockout strains. Mutants with the first phenotype were resistant to ciprofloxacin only and had the same substitution within GyrA as the NCTC11168-derived mutants. Irrespective of the parent strain, mutants with the second phenotype were resistant to ciprofloxacin, chloramphenicol, tetracycline, ethidium bromide, acridine orange, and SDS and had no mutation in gyrA. These mutants expressed levels of the efflux pump genes cmeB and cmeF and the major outer membrane protein gene porA similar to those expressed by the respective parent strains. No mutations were detected in cmeF or cmeB. Accumulation assays revealed that the mutants accumulated lower concentrations of drug. These data suggest the involvement of a non-CmeB or -CmeF efflux pump or reduced uptake conferring multiple-antibiotic resistance, which can be selected after exposure to a fluoroquinolone.

J Antimicrob Chemother. 2005 May;55(5):639-44. Epub 2005 Mar 10.

Characterization of antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea.

Kang HY, Jeong YS, Oh JY, Tae SH, Choi CH, Moon DC, Lee WK, Lee YC, Seol SY, Cho DT, Lee JC.

Department of Microbiology, Institute of Infectious Diseases and Department of Clinical Pathology, Kyungpook National University School of Medicine, 101 Dongin-dong, Jung-gu, Daegu 700-422, Korea.

OBJECTIVES: Antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea were characterized. METHODS: E. coli isolates were examined for susceptibility to antimicrobial agents. Integrase genes were amplified. Gene cassette regions for classes 1 and 2 integrons were amplified and sequenced. Conjugal transfer and Southern hybridization were performed to determine the genetic localization of class 1 integrons. The clonal relationship of E. coli isolates carrying an identical cassette array was analysed by PFGE. RESULTS: Commensal E. coli isolates from animals were highly resistant to commonly used antimicrobial agents such as tetracycline, sulfamethoxazole, streptomycin, ampicillin and carbenicillin. Integrons were most prevalent in commensal E. coli isolates from poultry (44%), followed by clinical isolates from humans (33%), commensal isolates from swine (23%) and humans (13%). dfrA17-aadA5, dfrA12-orfF-aadA2 and aadA1 were found most frequently in E. coli isolates from humans, poultry and swine, respectively. Class 1 integrons were mostly located in conjugative plasmids. E. coli isolates carrying an identical cassette array were phylogenetically unrelated. CONCLUSIONS: The use of antibiotics is strongly associated with antimicrobial resistance. E. coli isolates from different sources may select a specific gene cassette by antibiotic selective pressure, which results in differences in class 1 integrons. The horizontal transfer of class 1 integrons through conjugative plasmids seems to be responsible for wide dissemination of a particular type of class 1 integron.

J Antimicrob Chemother. 2005 May;55(5):776-9. Epub 2005 Mar 10.

Class 1 integron-associated gene cassettes in Salmonella enterica subsp. enterica serovar Agona isolated from pig carcasses in Brazil.

Michael GB, Cardoso M, Schwarz S. Institut fur Tierzucht, Bundesforschungsanstalt fur Landwirtschaft (FAL), Holtystr. 10, 31535 Neustadt-Mariensee, Germany.

OBJECTIVES: Two multiresistant Salmonella enterica subsp. enterica serovar Agona isolates from pig carcasses were investigated for antimicrobial resistance genes and their location with particular reference to the detection of class 1 integrons. METHODS: The two S. Agona isolates were investigated for their in vitro susceptibility to antimicrobial agents and their plasmid content. The resistance genes and class 1 amplicons were identified by PCR assays. Amplicons of class 1 integrons were cloned and sequenced. Transferability of resistance plasmids was confirmed by conjugation. RESULTS: Both S. Agona isolates carried conjugative plasmids of approximately 150 kb which harboured all resistance genes detected in the respective isolates. S. Agona 231 was resistant to chloramphenicol by catA1, to tetracycline and minocycline by tet(B), and to sulphonamides by sul1. In addition, it harboured a streptomycin resistance gene strA and a class 1 integron with a new aadA variant designated aadA23, which mediates resistance to streptomycin and spectinomycin. S. Agona 242 also carried the genes catA1, tet(B), and sul1. Moreover, it harboured a second sulphonamide resistance gene, sul2, and a class 1 integron with intact gene cassettes carrying new variants of the trimethoprim resistance gene dfrA15b or the chloramphenicol resistance gene cmlA4. The third gene cassette consisted of a truncated aadA2 gene. CONCLUSIONS: The results of this study show that large conjugative multiresistance plasmids are present in S. Agona from pigs. Analysis of the class 1 integrons revealed the presence of new variants of resistance genes so far not detected in Salmonella isolates.

[Editor's comments: I know you do not use FQs in pigs in the US but although we do in the UK we do not see a huge residual reservoir of FQ resistance in pigs as they are not given in drinking water (like poultry) or feed but as a piglet doser and by injection. By the time the pigs reach slaughter (National slaughter house survey 2003) resistance has subsided to negligible levels in E. coli (Nalidixic acid 1%, ciprofloxacin 0%) Salmonella enterica Typhimurium (NA 3%, Cip 0% (<1.0mcg/ml)) Campylobacter jejuni (NA 0%, Cip 0%) however in Campylobacter coli the results are higher (NA 27%, Cip 16%) demonstrating that there is a marked difference between the two campylobacter species, and suggesting that C. coli may have some inherent FQ resistance rather than just acquired.]